A phosphite dehydrogenase protein (PtxD) is a protein which exists in some bacteria and is an enzyme which oxidizes phosphorous acid in an NAD+-dependent or NADP+-dependent manner to generate NADH or NADPH. The following are reaction formulae respectively corresponding to a case where phosphorous acid is oxidized in an NAD+-dependent manner and a case where phosphorous acid is oxidized in an NADP+-dependent manner.
[Chem. 1]
                Catalyst: PtxDHPO32−+NAD++H2O→HPO42−+NADH+H+  (Reaction formula 1)[Chem. 2]        Catalyst: PtxDHPO32−+NADP++H2O→HPO42−+NADPH+H+  (Reaction formula 2)        
The above chemical reactions are expected as being highly potential because they allow efficient production of NADH or NADPH, each of which functions as a very important cofactor in production of a substance with use of a biological reaction. However, industrial use of the chemical reactions has not been developed yet. That is, at present, industrial mass-production of NADH or NADPH with use of phosphorous acid has not been realized (see, for example, Non-patent Literatures 1 and 2). Enzymes such as formic dehydrogenase, glucose dehydrogenase, alcohol dehydrogenase, and the like have conventionally been used for production of NADH and NADPH. However, since the enzymes use a highly reactive substrate, a reaction system thereof is unstable. Further, the enzymes yield a highly reactive product. This also makes the reaction system thereof unstable. Note, here, that a main cause for making the reaction system unstable is changes in pH. On the other hand, phosphite dehydrogenase is advantageous not only in that phosphite dehydrogenase uses a weakly reactive substrate and yields a weakly reactive product, but also in that the substrate used by phosphite dehydrogenase is inexpensive. Phosphite dehydrogenase therefore has a potential to be widely used in place of the above enzymes, if industrial use of phosphite dehydrogenase is realized. Specifically, in a case where phosphite dehydrogenase is used, a reaction system can be stabilized, since both phosphorous acid and phosphoric acid have a buffering action.
In order to produce NADH and NADPH in large quantities industrially, a large amount of the phosphite dehydrogenase protein is required. As such, it has conventionally been tried to obtain a large amount of the phosphite dehydrogenase protein by (i) forcibly causing expression of a heterologous organism-derived wild-type phosphite dehydrogenase protein in a host such as Escherichia coli and (ii) then purifying the wild-type phosphite dehydrogenase protein.
However, according to the technique, a large part of the wild-type phosphite dehydrogenase protein becomes insoluble in an aqueous solution when the wild-type phosphite dehydrogenase protein is forcibly expressed in E. coli or the like. This makes it impossible to collect the wild-type phosphite dehydrogenase protein. As such, there is a demand for a phosphite dehydrogenase protein which is highly soluble in an aqueous solution even in a case where the phosphite dehydrogenase protein is forcibly expressed.
Further, since a temperature of a reaction system rises in industrial production of NADH and NADPH in large quantities, it is necessary to use a phosphite dehydrogenase protein with high thermal stability. However, a conventional wild-type phosphite dehydrogenase protein has low thermal stability (specifically, many enzymes are denatured at 40° C.). As such, there is a demand for a phosphite dehydrogenase protein which can maintain high activity at high temperature.
Under such circumstances, it has been tried to screen for a phosphite dehydrogenase mutant which is improved in the above described properties.
For example, Non-patent Literature 3 discloses a technique in which a phosphite dehydrogenase protein, into which a mutation has been introduced, is forcibly expressed in E. coli, so that an amount of a mutated phosphite dehydrogenase protein contained in a soluble fraction is increased. Note that it cannot be determined from the data in Non-patent Literature 3 whether the increase in amount of the mutated phosphite dehydrogenase protein contained in the soluble fraction was caused by an increase in solubility of the mutated phosphite dehydrogenase protein or by an increase in expression level of the mutated phosphite dehydrogenase protein. Further, Non-patent Literatures 4 and 5 disclose mutated phosphite dehydrogenase proteins with high thermal stability.